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Tissue transglutaminase II (TGase II) is a dual function protein with both transamidating and guanidine triphosphate (GTP)-binding capabilities. Previous studies have implicated TGase as a pro-apoptotic molecule; however, our recent findings indicate that TGase II may act as a survival factor in various cell types. The purpose of this study was to survey TGase II expression in normal tissue and spontaneous tumours of dogs and cats, by Western blotting and immunohistochemistry. Bladder, liver and adrenal gland exhibited prominent expression of TGase II while other tissues, including mammary gland, displayed limited expression and activity. TGase II GTP-binding in normal tissues was proportional to the level of expression in all tissues examined. Normal mammary tissue and that showing benign hyperplasia did not express TGase II. However, 11/25 (44%) of canine mammary carcinomas and 10/12 (83%) of feline mammary carcinomas strongly expressed TGase II in either a stromal, cellular or combined pattern. The pattern of expression was not related to the classification of mammary carcinoma (solid, tubulopapillary, complex or anaplastic), except that two anaplastic canine mammary carcinomas showed prominent TGase II expression. Two canine mammary carcinoma cell lines showed prominent TGase expression, and when the TGase activity was inhibited, the cells became more sensitive to doxorubicin-induced cell death. Thus, TGase II was significantly expressed in mammary cancers from dogs and cats and immunoreactivity of TGase II was similar to that reported in humans beings. The pro-survival effect of TGase II in canine mammary carcinoma cell lines was similar to that previously reported in humans patients.

The human "Elston and Ellis grading method" was used in dogs with mammary carcinoma to examine its relation to prognosis in this species, based on a 2-year follow-up period. Of the 85 cases examined, 27(31.8%) had well-differentiated (grade I), 28 (32.9%) had moderately differentiated (grade II) and 30 (35.3%) had poorly differentiated (grade III) carcinomas. Two years after mastectomy, significant differences in survival between cases with different tumour grade were found; thus, survival was worse in dogs with grade III carcinomas than in those with grade II (P

Editor: We read with particular interest the 2007 article by Dr Chang et al, which describes the first case of canine mammary carcinoma with sebaceous differentiation. The authors compared the histologic features of the canine tumors with those of human cases. Since then, new and distinctive cases of this mammary carcinoma variant have been reported. Additionally, we sometimes encounter canine mammary gland carcinomas with sebaceous components among our own diagnostic cases. The aim of this letter is to draw the attention of Veterinary Pathology readers to the morphologic variation in reported mammary tumors with sebaceous components to complement the review by Dr Chang et al. Although it has been documented once in the veterinary literature, we believe that these additional data will contribute to the understanding of this peculiar mammary carcinoma subtype. Although the number of well-documented cases is still limited, the World Health Organization classification for human breast tumors now recognizes sebaceous carcinoma as a distinct subtype of invasive breast carcinoma. However, the presence of sebaceous differentiation and other neoplastic components vary markedly among the reported cases, which makes the classification criteria of mammary sebaceous carcinoma ambiguous. A sebaceous component has been described in mammary adenoid cystic carcinomas, invasive ductal carcinoma (some with in situ components of cribriform and comedocarcinoma types), and intraductal breast papilloma with squamous/apocrine metaplasia. Other authors have described tumors with a predominance of sebaceous cells (> 50%), with or without basal cells, and have thus classified them as primary sebaceous carcinomas of the breast. Dr Chang and colleagues were prudent in classifying their canine case as a mammary carcinoma with sebaceous differentiation because, apparently, some cells of the typical ductal carcinoma component had undergone sebaceous differentiation. However, because extensive solid multilobulated neoplastic tissue composed of basal and sebaceous cells was also observed, they probably could have used the term mammary sebaceous carcinoma due to the predominance of sebocytic cells in the neoplasm. The morphologic characteristics (and the degree of cellular atypia) of the nonsebaceous components in these mammary carcinomas may be quite variable. Thus, we believe that the term sebaceous carcinoma is not suitable for all of these lesions and that care must be taken when evaluating mammary tumors with sebaceous differentiation because the nonsebaceous component may be quite aggressive. Histochemistry is helpful in the diagnosis of these neoplasms. The lipid vacuoles in the sebocytic cells are not stained by periodic acid–Schiff, mucicarmine, or alcian blue but are stained by oil red O. Additionally, immunohistochemistry to detect adipophilin may be performed to support the diagnosis. Whereas the oil red O technique is not reliable for detection of lipid in paraffin-embedded tissue, the antibodies to adipophilin will bind sebaceous cells, even in tissues that were routinely processed with the lipid solvents used in paraffin embedding. The origin of the sebaceous component in these mammary carcinomas is unknown. Some authors suggest that basaloid cells of the tumors can differentiate toward cutaneous adnexal structures or that local stem cells might give rise to sebocytes.

Background Tumor-associated macrophages (TAMs) have high impact on the cancer development because they can facilitate matrix invasion, angiogenesis, and tumor cell motility. It gives cancer cells the capacity to invade normal tissues and metastasize. The signaling of colony-stimulating factor-1 receptor (CSF-1R) which is an important regulator of proliferation and differentiation of monocytes and macrophages regulates most of the tissue macrophages. However, CSF-1R is expressed also in breast epithelial tissue during some physiological stages i.g.: pregnancy and lactation. Its expression has been also detected in various cancers. Our previous study has showed the expression of CSF-1R in all examined canine mammary tumors. Moreover, it strongly correlated with grade of malignancy and ability to metastasis. This study was therefore designed to characterize the role of CSF-1R in canine mammary cancer cells proliferation, apoptosis, migration, and invasion. As far as we know, the study presented hereby is a pioneering experiment in this field of veterinary medicine. Results We showed that csf-1r silencing significantly increased apoptosis (Annexin V test), decreased proliferation (measured as Ki67 expression) and decreased migration (“wound healing” assay) of canine mammary cancer cells. Treatment of these cells with CSF-1 caused opposite effect. Moreover, csf-1r knock-down changed growth characteristics of highly invasive cell lines on Matrigel matrix, and significantly decreased the ability of these cells to invade matrix. CSF-1 treatment increased invasion of cancer cells. Conclusion The evidence of the expression and functional role of the CSF-1R in canine mammary cancer cells indicate that CSF-1R targeting may be a good therapeutic approach.

Solid tumors comprise of various cells, including cancer cells, resident stromal cells, migratory haemopoietic cells and other. These cells regulate tumor growth and metastasis. Macrophages constitute probably the most important element of all interactions within the tumor microenvironment. However, the molecular mechanism, that guides tumor environment, still remains unknown. Exploring the underlying molecular mechanisms that orchestrate these phenomena has been the aim of our study. The co-cultures of five various canine mammary carcinoma cell lines and macrophages were established and maintained for 72 hrs. After that, having sorted the cells, an integrated analysis of genome-wide mRNA and microRNA expression profiles have been assessed. The analysis showed that the up-regulated genes in the cancer cell lines grown as co-culture with macrophages are involved mainly in: macrophages activation, cell motion, mammary gland development, cell-cell adhesion, and angiogenesis. Basides, the presence of macrophages in the cancer environment induces acquisition of the macrophage antigens and phenotype in cancer cells. The up-regulated genes in macrophages grown as co-cultre with cancer cells were involved mainly in cell-cell adhesion and cell-matrix adhesion. We found that co-culture of cancer cells and macrophages regulates expression of several important miRNAs. Taken together, this integrated comparative study generated a scheme of the molecular changes that occur during interactions between macrophage and cancer. Because as far as we realize this is the first analysis of changes in miRNA expression in cancer cells due to a presence of macrophages, our study can serve as a valuable source for future studies on a tumor microenvironment, and some of the highlighted genes, miRNAs, pathways or processes may be useful for diagnostic or therapeutic purposes. This work was supported by grant no N N308012939 from the Ministry of Sciences and Higher Education. Citation Format: Magdalena Król, Karol Pawlowski, Kinga Majchrzak, Alicja Majewska, Tomasz Motyl. Comparative mRNA and microRNA expression profiling of canine mammary carcinoma cell lines and macrophages grown as a co-culture in vitro. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A76.

Two murine monoclonal antibodies (MAbs), 1A10 and SB2, generated against a canine mammary carcinoma cell line, were used in a competitive enzyme-linked immunosorbent assay (ELISA) to measure tumor-associated antigens (TAAs) in canine serum samples. Sera were tested from disease-free dogs and from dogs diagnosed with mammary carcinoma, non-mammary carcinoma, sarcoma, benign mammary tumor, benign non-mammary tumor, or non-neoplastic disease. Serum antigen concentrations measured by ELISA were expressed as inhibitory units (IU). The upper limit of normal, defined as the mean plus 2 SD of the TAA concentration in disease-free dogs, was 20 IU with antibody 1A10 and 22 IU with antibody SB2. Compared with disease-free dogs, the frequency of TAA-positive sera was significantly greater (P < 0.05) among dogs with mammary or non-mammary carcinoma when tested with MAbs 1A10 or SB2, and also with sarcoma when tested with MAb SB2. Testing a serum sample with both antibodies rather than just one increased the sensitivity of the competitive ELISA for TAA detection. The presence of TAA in serum might serve as a useful marker for certain types of carcinomas or sarcomas in canine cancer patients.

In human medicine, passive immunotherapy is a well-established tool to fight cancer. Cetuximab and trastuzumab are famous examples of effective monoclonal antibody therapies for the treatment of ErbB-1 (EGFR) overexpressing colon tumors and for ErbB-2 (HER2/neu) overexpressing metastatic breast cancer. Mammary carcinomas of dogs are the most important cause of tumor-related death in females. It is known that these tumors also express tumor markers similar to human breast cancer and that regarding the idea of comparative oncology cancer in pet dogs are a valuable model for speeding up drug development. Therefore, we aimed to investigate more closely canine ErbB-1 and ErbB-2 and the applicability of cetuximab and trastuzumab in the dog. Screenings with FDA-approved immunohistochemical diagnostic tests of canine mammary tumor samples showed 3/10 ErbB-1 and 4/10 ErbB-2 overexpression, which seems similar to the expression pattern in human breast cancer patients. Sequence analyses revealed amino acid sequence identities of 91% between human and canine ErbB-1 and 92% for ErbB-2. Modeling of those proteins showed that the human cetuximab epitope differs in only four amino acids compared to the canine counterpart and that the trastuzumab binding site is identical in human and canine ErbB-2 except for only one amino acid. Using flow cytometry analyses cetuximab and trastuzumab binding could be confirmed in four canine mammary carcinoma cell lines. Furthermore, cell viability assays showed that incubation of canine cell lines with both antibodies inhibited tumor cell proliferation, even though there are lower numbers of ErbB molecules on canine than on human cells, which was confirmed by a flow cytometry-based assay. This comparative oncology study indicates that ErbB-1 and ErbB-2 are highly conserved molecules with significant concordance in terms of structure and expression pattern between human and canine cancer. Importantly, the canine homologous molecules are susceptible to cetuximab and trastuzumab targeting, which may open up new avenues towards antibody-based immunotherapies in companion dogs and improvements in anticancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C46.

Background Similar to human breast cancer mammary tumors of the female dog are commonly associated with a fatal outcome due to the development of distant metastases. However, the molecular defects leading to metastasis are largely unknown and the value of canine mammary carcinoma as a model for human breast cancer is unclear. In this study, we analyzed the gene expression signatures associated with mammary tumor metastasis and asked for parallels with the human equivalent. Methods Messenger RNA expression profiles of twenty-seven lymph node metastasis positive or negative canine mammary carcinomas were established by microarray analysis. Differentially expressed genes were functionally characterized and associated with molecular pathways. The findings were also correlated with published data on human breast cancer. Results Metastatic canine mammary carcinomas had 1,011 significantly differentially expressed genes when compared to non-metastatic carcinomas. Metastatic carcinomas had a significant up-regulation of genes associated with cell cycle regulation, matrix modulation, protein folding and proteasomal degradation whereas cell differentiation genes, growth factor pathway genes and regulators of actin organization were significantly down-regulated. Interestingly, 265 of the 1,011 differentially expressed canine genes are also related to human breast cancer and, vice versa, parts of a human prognostic gene signature were identified in the expression profiles of the metastatic canine tumors. Conclusions Metastatic canine mammary carcinomas can be discriminated from non-metastatic carcinomas by their gene expression profiles. More than one third of the differentially expressed genes are also described of relevance for human breast cancer. Many of the differentially expressed genes are linked to functions and pathways which appear to be relevant for the induction and maintenance of metastatic progression and may represent new therapeutic targets. Furthermore, dogs are in some aspects suitable as a translational model for human breast tumors in order to identify prognostic molecular signatures and potential therapeutic targets.

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Tumours were induced in CBA athymic nude mice by subcutaneous injection of REM 134 cells. These cells were from a continuous line derived from a canine mammary carcinoma and have no detectable oestrogen receptors. In vitro, the anti-oestrogen tamoxifen was growth inhibitory at a concentration of 10(-6) M and adding 10(-8) M oestradiol-17 beta did not reverse this effect. The relative rate of growth of the tumours induced by the cultured cells was the same in male and female mice. Oral tamoxifen at a dose of 1 mg mouse-1 week-1 suppressed the early growth rate significantly and to the same extent in male and female mice; conversely subcutaneous tamoxifen at 2 mg mouse-1 week-1 had only a transient early effect. These results argue against tamoxifen acting solely as an antagonist for oestrogen.

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Hybridoma-derived murine monoclonal antibodies (MoAbs) were generated by fusing P3X63-Ag8.653 myeloma cells with splenic cells from BALB/c mouse which had been immunized with viable canine mammary adenocarcinoma cells, CMT-2. Fifteen MoAbs were shown to react with immunizing cells in indirect immunofluorescence (IFA) and enzyme-linked immunosorbent (ELISA) assays. The reactivity of one IgM MoAb, designated 4A9, was evaluated. The antigen recognized by 4A9 on CMT-2 cells appeared to be localized both in cell membrane and cytoplasm against fixed and unfixed preparations by IFA. The 4A9 MoAb was found to bind with four of five canine mammary carcinoma cell lines while no binding was detected with normal fibroblastic cell lines. In vivo tissue distribution of 4A9 antigen was evaluated by indirect immunoperoxidase (IP) assay against formalin-fixed, paraffin-embedded sections of normal and neoplastic tissues. 4A9 MoAb reacted strongly to moderately with 75% of mammary carcinomas, moderately to weakly with 57% of benign mammary tumors, and strongly with squamous cell and perianal gland carcinomas (100%), interstitial cell tumors (100%), transitional cell carcinomas (43%), lung adenocarcinomas (40%), colon carcinomas (33%), and pancreatic adenocarcinomas (20%). Moderate to weak staining was detected with granulosa cell tumors (25%) and apocrine gland adenocarcinomas (50%). Strong reactivity with perianal gland carcinomas contrasted to no reactivity with perianal gland adenomas. No immunostaining was detected with a large variety and number of normal adult and fetal tissues tested; negligible and very restricted staining was observed in a few adult and fetal tissues. Normal mammary gland was negative. Since the antigen is expressed on the cell surface and in the cytoplasm of most mammary carcinoma cells and a variety of other epithelial tumor cells, the 4A9 antibody may have potential application in diagnosis and management of canine mammary cancer and a variety of other epithelial tumors.

Many of the spontaneous malignant tumours which occur in the dog have a similar behaviour to their counterparts in man and are very valuable in comparative cancer studies. World Health Organisation international clinical trials on therapy of canine osteosarcoma, lymphosarcoma and mammary carcinoma, which are good ‘models’ for these conditions in man, are planned. There are, however, certain observations and investigations which can be better performed on dogs with transplanted tumours than in these clinical cases, and suitable methods of doing this in an outbred species have been achieved.

The main criteria for spontaneous cancer models are:— 1. The histological type of tumour should resemble the human tumour. 2. The biological behaviour, particularly growth rate and metastasis, should be similar. 3. The size of the animal should permit the required clinical investigations. 4. The tumour incidence should be high enough to study a large number of cases.

Three cells lines, REM 134, 111 and 367, have been derived from canine mammary carcinomas and their morphological characteristics in vitro are described. They are tumorigenic in athymic nude mice, have no demonstrable fibronectin on their cell surfaces and exhibit a varied pattern of lectin binding. They can be cloned in semi-solid agar. One line, REM 134, responds to oestrogen and luteotropic hormone in vitro, although none of the three had demonstrable oestrogen receptors.

We studied 232 dogs that underwent mastectomy for mammary epithelial neoplasms. The mastectomy specimens were evaluated according to structural parameters found to be prognostically significant in human mammary neoplasms, such as grade of atypia in non-invasive proliferations of duct epithelium, extent of malignant disease, and nuclear differentiation in malignant neoplasms. In this study, the biologic behavior of mammary lesions was assessed according to the frequency of development of de novo or recurrent invasive carcinoma within two years. Our data indicate that we can recognize structural variables which permit classification of mammary neoplasms into categories with distinct patterns of biologic behavior. In addition to normotypic proliferative lesions and invasive malignant neoplasms, we were able to identify precancerous atypic and non-invasive malignant neoplasms that are considered precursor lesions in women. Also, as in women, nuclear differentiation was found to be a prognostically significant variable. Lymphoid cellular reactions, considered to be structural correlates of host-tumor immune responses in women, were noted in 35% of dogs with precancerous or malignant neoplasms. Application of the described parameters should facilitate comparative studies of canine and human mammary carcinogenesis and use of the dog as a model for the development of new therapeutic modalities and immunoprophylaxis of human mammary cancer.

Twenty-three of 27 canine mammary tumours grew in culture following overnight trypsination at room temperature. A solid carcinoma and a tubular adenocarcinoma are probable cell lines. The adenocarcinoma appeared fibroblastic in tissue culture but when transplanted into an immunosuppressed dog produced an adenocarcinoma similar to that in the original dog.

Targeting mitochondrial energy metabolism is a novel approach in cancer research and can be traced back to the description of the Warburg effect. Dichloroacetate, a controversially discussed subject of many studies in cancer research, is a pyruvate dehydrogenase kinase inhibitor. Dichloroacetate causes metabolic changes in cancerous glycolysis towards oxidative phosphorylation via indirect activation of pyruvate dehydrogenase in mitochondria. Canine mammary cancer is frequently diagnosed but after therapy prognosis still remains poor. In this study, canine mammary carcinoma, adenoma and non-neoplastic mammary gland cell lines were treated using 10 mM Dichloroacetate. The effect on cell number, lactate release and PDH expression and cell respiration was investigated. Further, the effect on apoptosis and several apoptotic proteins, proliferation, and microRNA expression was evaluated. Dichloroacetate was found to reduce cell proliferation without inducing apoptosis in all examined cell lines.

Background: Current studies report that aberrations in epigenetic regulators or chromatin modifications are related to tumor development and maintenance. EZH2 (Enhancer of zeste homolog 2) is one of the catalytic subunits of Polycomb repressive complex 2, a crucial epigenetic regulator. EZH2 has a master regulatory function in such processes as cell proliferation, stem cell differentiation, and early embryogenesis. In humans, EZH2 is linked to oncogenic function in several carcinomas, including breast cancer, and dysregulation of EZH2 has been particularly associated with loss of differentiation and the development of poorly differentiated breast cancer. In our present study, we were interested in determining whether EZH2 is increased in canine mammary tumors, which show similarities to human breast cancer. Results: Investigation of the expression of EZH2 in canine mammary tumors revealed that EZH2 protein was overexpressed in canine mammary carcinomas, as in human breast cancer. In addition, the immunohistochemical expression level of EZH2 was associated with the degree of malignancy in canine mammary carcinoma. This is the first report to describe EZH2 expression in canine mammary tumors. Conclusions: Because the expression of EZH2 was similar in canine mammary carcinoma and human breast cancer, spontaneous canine mammary tumors may be a suitable model for studying EZH2 and treatment development.

Background Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. Methods Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. Results We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. Conclusions Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1235-8) contains supplementary material, which is available to authorized users.

Background Canine mammary carcinoma is the most common cancer in female dogs and is often fatal due to the development of distance metastasis. The microenvironment of a tumour often contains abundant infiltrates of macrophages called tumour-associated macrophages (TAMs). TAMs express an activated phenotype, termed M2, which sustains proliferation of cancer cells, and has been correlated with poor clinical outcomes in human cancer patients. Cancer cells themselves have been implicated in stimulating the conversion of macrophages to a TAM with an M2 phenotype. This process has yet to be fully elucidated. Here we investigate the interplay between cancer cells and macrophages in the context of canine mammary carcinoma. Results We show that cancer cells inhibit lipopolysaccharide (LPS)-induced macrophage activation. Further, we show that macrophage associated proteins, colony-stimulating factor (CSF)-1 and C-C motif ligand (CCL)-2, stimulate macrophages and are responsible for the effects of cancer cells on macrophages. We suggest the existence of a feedback loop between macrophages and cancer cells; while cancer cells influence the phenotype of the TAMs through CSF-1 and CCL2, the macrophages induce canine mammary cancer cells to upregulate their own expression of the receptors for CSF-1 and CCL2 and increase the cancer cellular metabolic activity. However, these cytokines in isolation induce a phenotypic state in macrophages that is between M1 and M2 phenotypes. Conclusions Overall, our results demonstrate the extent to which canine mammary carcinoma cells influence the macrophage phenotype and the relevance of a feedback loop between these cells, involving CSF-1 and CCL2 as important mediators. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0473-y) contains supplementary material, which is available to authorized users.

Canine mammary carcinoma (CMC) has been used as a model to investigate the pathogenesis of human breast cancer and the same grading scheme is commonly used to assess tumor malignancy in both. One key component of this grading scheme is the density of mitotic figures (MF). Current publicly available datasets on human breast cancer only provide annotations for small subsets of whole slide images (WSIs). We present a novel dataset of 21 WSIs of CMC completely annotated for MF. For this, a pathologist screened all WSIs for potential MF and structures with a similar appearance. A second expert blindly assigned labels, and for non-matching labels, a third expert assigned the final labels. Additionally, we used machine learning to identify previously undetected MF. Finally, we performed representation learning and two-dimensional projection to further increase the consistency of the annotations. Our dataset consists of 13,907 MF and 36,379 hard negatives. We achieved a mean F1-score of 0.791 on the test set and of up to 0.696 on a human breast cancer dataset.

// Judit Fazekas-Singer 1, 2 , Neydher Berroteran-Infante 3, 4 , Christina Rami-Mark 2, 3, 4 , Monika Dumanic 3 , Miroslawa Matz 2 , Michael Willmann 5 , Fritz Andreae 6 , Josef Singer 2, 3, 7 , Wolfgang Wadsak 3, 4, 8 , Markus Mitterhauser 3, 9 and Erika Jensen-Jarolim 1, 2 1 Comparative Medicine, The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna, and University of Vienna, Vienna, Austria 2 Institute of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria 3 Department of Biomedical Imaging and Image-Guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Vienna, Austria 4 Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria 5 Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria 6 piCHEM Forschungs- und Entwicklungs GmbH, Graz, Austria 7 Department of Internal Medicine II, University Hospital Krems, Karl Landsteiner University of Health Sciences, Krems an der Donau, Austria 8 CBmed GmbH-Center for Biomarker Research in Medicine, Graz, Austria 9 LBI Applied Diagnostics, Vienna, Austria Correspondence to: Erika Jensen-Jarolim, email: erika.jensen-jarolim@meduniwien.ac.at Keywords: epidermal growth factor receptor, radio-immunotherapy, canine mammary carcinoma, canine antibody, comparative oncology Received: August 02, 2017 Accepted: August 23, 2017 Published: September 15, 2017 ABSTRACT Due to large homology of human and canine EGFR, dogs suffering from spontaneous EGFR+ cancer can be considered as ideal translational models. Thereby, novel immunotherapeutic compounds can be developed for both human and veterinary patients. This study describes the radiolabeling of a canine anti-EGFR IgG antibody (can225IgG) with potential diagnostic and therapeutic value in comparative clinical settings. Can225IgG was functionalized with DTPA for subsequent chelation with the radionuclide 99m Tc. Successful coupling of 10 DTPA molecules per antibody on average was proven by significant mass increase in MALDI-TOF spectroscopy, gel electrophoresis and immunoblots. Following functionalization and radiolabeling, 99m Tc-DTPA-can225IgG fully retained its binding capacity towards human and canine EGFR in flow cytometry, immuno- and radioblots, and autoradiography. The affinity of radiolabeled can225IgG was determined to K D 0.8 ±0.0031 nM in a real-time kinetics assay on canine carcinoma cells by a competition binding technique. Stability tests of the radiolabeled compound identified TRIS buffered saline as the ideal formulation for short-term storage with 87.11 ±6.04% intact compound being still detected 60 minutes post radiolabeling. High stability, specificity and EGFR binding affinity pinpoint towards 99m Tc-radiolabeled can225IgG antibody as an ideal lead compound for the first proof-of-concept diagnostic and therapeutic applications in canine cancer patients.

Background Krüppel-like factors (KLFs) are critical regulators of biological and physiological systems and have been extensively studied for their roles in cell proliferation, differentiation and survival in the context of cancer. Among the KLFs, KLF4 is highly expressed in human breast cancers and plays an oncogenic role. The present study examined the expression of KLF4 and assessed its significance in canine mammary carcinoma. Results Immunohistochemistry was employed to investigate the expression of KLF4 in 142 cases of canine mammary tumor. 75 of the 142 (52.8%) cases were histologically confirmed as mammary carcinoma. Quantification of immunohistochemistry was carried out using Quick score which multiply the staining intensity by the percentage of positive cells. High KLF4 expression was identified in 44 of the 75 (59%) dogs with mammary carcinoma and none in the benign cases. High KLF4 expression occurred only in the tumor cells and not the adjacent normal cells in mammary carcinoma (P < 0.001). Moreover, the high expression level of KLF4 expression was statistically associated with poor grade, late stage, histological subtypes of simple and complex carcinoma, and shorter 24-month survival. The Kaplan-Meier survival analysis also indicated that dogs with high nuclear KLF4 expression had a significantly shorter survival than those with low/moderate KLF4 expression (P = 0.011). Conclusions KLF4 is highly and frequently expressed in canine mammary carcinoma and correlates with a more aggressive phenotype.